A number of previous pre-clinical studies suggest that anti-angiogenic agents create normalization window during which delivery and efficacy of concurrently administered cytotoxic agents is enhanced. However, recent studies have shown that vessel normalization results in the restoration of blood brain barrier and consequential inefficient vessel dependent delivery of cytotoxic drugs to brain tumors. These results have raised fundamental questions about modes of delivery and the types of anti-angiogenic and cytotoxic drug combinations used to 1) normalize tumor vasculature; and 2) enhance the outcome of cytotoxic therapy post-normalization for brain tumors? This has prompted the design of novel therapies that allow use of 1) alternative modes of drug delivery that targets both the primary tumors and secondary micro-invasive deposits in the brain; 2) engineered therapeutic proteins that specifically target both tumor cells and associated vasculature; and 3) diagnostic proteins that allow tracking of drug delivery vehicles, therapeutic proteins and fate of tumors in vivo. In this proposal, we will engineer human neural stem cells (NSC) with secretable antiangiogenic (aa), thrombospondin (TSP)-1 and pro-apoptotic secretable tumor necrosis factor apoptosis inducing ligand (S-TRAIL) which is known to induce apoptosis via death receptor (DR)4/5 and initially compare the normalization potential of NSC-aaTSP-1 with systemic delivery of known anti-angiogenic agents in established malignant and primary invasive brain tumors (gliomas). Based on the recent studies by our Co-Investigator, Dr. Lawler, that TSP-1 normalizes vasculature and also induces death receptor (DR)4/5 expression on tumor associated endothelial cells (EC) priming them to TRAIL-induced killing, the therapeutic effect of S-TRAIL post vessel normalization and EC priming by aaTSP-1 will be tested in both malignant and invasive glioma models. We hypothesize that aaTSP-1 will enhance the cytotoxic effects of S-TRAIL on tumor cells by normalizing the vasculature and also upregulation of DR4/5 expression in EC. This will ultimately lead to enhanced eradication of tumor cells and also killing of EC in both the primary tumor mass and the micro-invasive tumor cells escaping the primary tumor mass. A close integration of fluorescent and bioluminescent imaging markers into glioma cells, EC and NSC will allow us to assess therapeutic efficacy early and quickly and thus to adjust and fine-tune the proposed therapeutic approaches. In an effort to simulate a clinical scenario, the therapeutic NSC will be encapsulated into biocompatible synthetic extracellular matrix (sECMs) and tested in our recently developed resection model of glioma. Once validated, we envision a therapeutic modality in which at the time of brain tumor surgery, the main tumor mass will be removed and genetically engineered therapeutic cells will be introduced in sECMs and allowed to target the remaining tumor cells and micro-invasive tumor deposits in the brain. This will have a major impact in saving the lives of many brain cancer patients.